A new ent-kaurane diterpenoid from Ixora amplexicaulis

A new ent-kaurane diterpenoid, 6α,16α-dihyroxy-ent-kaurane (1), was isolated from the stems of Ixora amplexicaulis, together with (24R)-6β-hydroxy-24-ethyl-cholest-4-en-3-one (2), 7β-hydroxysitosterol (3), maslinic acid (4), 3,3′-bis(3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) (5) and protocatechuric acid (6). Their structures were established by extensive spectroscopic analysis, including 2D NMR techniques. Compounds 2–5 were isolated from the genus Ixora for the first time and 6 obtained originally from I. amplexicaulis.


Introduction
Ixora is a genus of about 400 species in the family Rubiaceae, mainly distributing in tropical Asia, Africa and Oceania, with 19 species being endemic to South China (Lo et al. 1999). The plants of the genus usually have small beautiful flowers, being easily cultured as common roadside and garden trees. The leaves, flowers, stems and roots of the genus have also been used in traditional medicine to treat various ailments. For example, the leaves and stems of Ixora coccinea are widely used in traditional Sudanese and Ayurvedic medicinal systems for the treatment of diarrhoea, fever, headache, skin diseases, eye trouble, wounds, sores and ulcers (Jaiswal et al. 2014). Previous investigations led to the isolation of many compounds including phenolics, peptides, terpenoids and sterols from plants of the genus (Ragasa et al. 2004;Idowu et al. 2010;Lee et al. 2010;Ikram et al. 2013). Pharmacological studies showed that extracts of the genus and the isolated compounds displayed extensive activities, such as antitumour, chemoprotective and antioxidant activities (Latha & Panikkar 1998;Wen et al. 2011;Wickramasinghe et al. 2014). But there was no report on Ixora amplexicaulis, which is a small shrub endemic to China. As a part of our research of structurally unique and biologically active compounds from medicinal plants of Yunnan, China, we have isolated and identified a new entkaurane diterpenoid, 6a,16a-dihyroxy-ent-kaurane (1), as well as five known compounds, (24R)-6b-hydroxy-24-ethyl-cholest-4-en-3-one (2), 7b-hydroxysitosterol (3), maslinic acid (4), 3,3 0 -bis(3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) (5) and protocatechuric acid (6) from the stems of I. amplexicaulis (Figure 1). Compounds 2 -5 were isolated from the genus Ixora for the first time and 6 obtained originally from I. amplexicaulis.

Apparatus and reagents
Optical rotations were determined on a Horiba SEAP-300 spectropolarimeter. IR was measured on a Perkin-Elmer 241 polarimeter. Mass spectrometry (MS) was obtained on a VG Auto Spec-3000 spectrometer. NMR spectra were recorded on a Bruker DRX-400 spectrometer at 400 MHz for 1 H and 100 MHz for 13 C using standard pulse sequence programs. All chemical shifts were recorded with respect to tetramethylsilane (TMS) as an internal standard. Column chromatography was carried out on silica gel (200 -300 mesh, Qingdao Marine Chemical Co., Qingdao, China), MCI-gel CHP 20P (75 -150 mm, Mitsubishi Chemical Corp., Tokyo, Japan) and Fuji Chemical Industrial Co.,Ltd,Tochigi,Japan). Thin layer chromatography (TLC) was performed on silica gel GF 254 (Yantai Jiangyou Silica Gel Co. Ltd, Yantai, China). Solvents were of industrial purity and distilled before use.

Plant material
The stems of I. amplexicaulis were collected from Mengla County of Yunnan Province, China in June, 2006 and identified by Mr Chaozhong Peng, a botanist of Yunnan Branch, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, where a voucher specimen (no. 0606005) was deposited.

Conclusion
In our research, chemical constituents of I. amplexicaulis were first investigated and six compounds were isolated from the stems of I. amplexicaulis. 6a,16a-dihyroxy-ent-kaurane (1) is a new ent-kaurane diterpenoid. Compounds 2 -5 were isolated from the genus Ixora for the first time and 6 obtained originally from I. amplexicaulis.

Supplementary material
Supplementary material relating to this article is available online.